The flavivirus family includes several clinically important animal viruses, including Dengue, West Nile, Japanese encephalitis, yellow fever, and tick-borne encephalitis viruses. Dengue is one of the most serious infectious diseases globally. There are about 100 million cases every year, with over 500,000 cases of potentially fatal Dengue hemorrhagic fever. Dengue virus (DENV) puts nearly 2.5 billion people at risk of infection in tropical and subtropical countries. Similarly, West Nile virus (WNV) has caused thousands of human infections in North America, besides infecting people on other continents. WNV infection can lead to serious illnesses in humans, resulting in encephalitis and death. Neither a prophylactic vaccine nor antiviral therapies are available for both WNV and DENV. The development of either a vaccine or an antiviral drug requires detailed knowledge of the viral life cycle.
The flaviviruses have a small positive sense RNA genome that is translated into a polyprotein, which is co- and post-translationally cleaved by both viral and host proteases into three structural (C, E, and M) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The non-structural proteins aid in viral genome replication. Since viruses encode a limited number of proteins, it is likely that viral proteins recruit or interact with several host proteins to make the cellular environment conducive to viral replication by inhibiting or interfering with the function of cellular factors that would otherwise obstruct virus infection and production. Several investigators have successfully utilized high-throughput screening methods (e.g., a genome- scale RNAi screen) to identify the mammalian or insect host genes that either facilitate or interfere with viral replication. These proteins are involved in various host cell processes, including intracellular protein trafficking, signal transduction, ion and molecular transport, and nucleic acid, protein, and lipid metabolism. Recent protein—protein interaction studies between viral proteins and host factors indicated that each viral protein interacts with several host proteins, suggesting that each viral protein performs multiple functions by interacting or recruiting different host factors.
The RNAi- screening studies and several proteomic studies have identified a role of the ubiquitin- proteasome system (UPS) for WNV and DENV replication Inhibition of the UPS by RNAi or by a chemical inhibitor significantly reduces the viral yield. However, the targets of the UPS are currently unknown and require further investigation. The UPS is a major extralysosomal protein- degradation pathway that degrades misfolded or unnecessary proteins from the cytosol and the nucleus, and also provides for signal-dependent or temporally specific degradation of numerous regulatory proteins. It plays a key role in maintaining cellular protein homeostasis. Consequently, it is involved in several cellular processes, including the stress response, cell-cycle regulation, DNA repair, antigen presentation, apoptosis, signal transduction, and transcriptional regulation. Proteins destined for degradation by the UPS are tagged with ubiquitin in a cascade of reactions, involving ubiquitin activation by a ubiquitin-activating enzyme (E1), followed by transfer of the activated ubiquitin to a ubiquitin-conjugating enzyme (E2). Finally, the ubiquitin-protein ligase (E3) transfers the ubiquitin to the target protein to an internal lysine residue on the substrate. After the initial ubiquitination event on a given substrate, additional ubiquitin groups are often added, and in many cases the additional ubiquitin groups are attached to a previously conjugated ubiquitin rather than directly to the substrate. This type of ubiquitin conjugation leads to the formation of “ubiquitin chains” on substrates. Once the target protein is tagged with ubiquitin, it is then degraded by the 26S proteasome protein complex with the release of ubiquitin for recycling. The efficiency of targeting a substrate for degradation is thought to depend on the number of bound ubiquitin groups, or the length of the bound ubiquitin chains, with chains of four or more ubiquitin groups allowing for rapid substrate turnover.
Ubiquitination is a reversible process; the ubiquitin chain can be made shorter or removed by a set of enzymes known as deubiquitinating enzymes (DUBs). Modification of the ubiquitin chain length regulates substrate degradation rates by altering substrate affinity for the proteasome. Ubiquitin-specific proteases (USPs) and ubiquitin C-terminal hydrolases (UCHs) are the best-characterized DUBs. Several DUBs have been implicated in disease mechanisms, including neurological disorders, infectious diseases, and cancer. Consequently, DUBs are plausible targets for drug discovery, and several small-molecule inhibitors targeting DUBs have been identified. Recently, a small molecule inhibitor of a mammalian proteasome—associated DUB, USP14, was identified. The compound 1-[1-(4-fluorophenyl)-2,5-dimethylpyrrol-3-yl]-2pyrrolidin-1-ylethanone (also called IU1) is specific for USP14, and it enhances proteolysis in mammalian cells.
Members of multiple families of both RNA and DNA viruses reprogram the UPS for various purposes, including immune evasion, viral entry and release, transcriptional regulation, and apoptosis. For example, human cytomegalovirus, herpesviruses, and Epstein Barr virus escape host immune responses by altering the processing of MHC molecules by the proteasome. Retroviruses require proteasome activity for processing of the Gag protein for efficient release of viral progeny. Transcriptional activation of herpesvirus VP16 also requires proteasome activity. Human papilloma virus E6 protein interacts with ubiquitin ligase E6-associated protein and target p53 for degradation, preventing apoptosis. The UPS facilitates entry of influenza virus into host cells. Inhibition of proteasome activity markedly reduces coxsackievirus (CVB3) RNA and protein levels.
In general, inhibiting USP14 is not toxic to cells, nor are USP14-null murine embryonic fibroblasts compromised in viability. There exists a need for antiviral therapies that are not detrimental to host cell viability.